Good Correlation Between Amplified and Non-Amplified RNA

For any method of gene transcript amplification method, it is important that the relative levels of RNAs be maintained during the amplification process. A quantitative comparison of the levels of small RNA before and after amplification is shown in Fig 3. In this experiment, 50 ng of a pool of RNAs from 4 different tissues was amplified with the SBI kit and tested for the presence of 10 different noncoding small RNA by RT-PCR.

The same RNA pool was tested and quantitated for the same small noncoding RNA before amplification. Care was taken to avoid the plateau phase of the PCR amplification.

The results are shown in Fig. 3. For all 10 small RNAs, the relative levels were maintained before and after amplification.

 

Size Distribution of Amplified cDNA

Global amplification of small RNAs. The size distribution and banding pattern of amplified cDNA is shown in Figure 2. In this study, 50 ng of a pool of RNAs from 4 different tissues was amplified with the SBI kit and a small portion examined by gel electrophoresis. The results are shown in Figure 2.

 

A banding pattern was observed which is consistent with amplification of several highly abundant small RNAs, as indicated. Because of the very small size and heterogeneity of microRNAs (19-24 nucleotides), and low abundance, it is not possible to observe the microRNA. The presence of microRNA is clearly visible. The yield of amplified cDNA is sufficient for several hundred quantitative PCR experiments or several microRNA microarray experiments. Note that the sizes of the microRNA amplified species (including the 60 nt adaptors) would range from 79 to 84 nts.

High Representation of Small RNAs After Amplification

Example of the representation of small noncoding RNA following RNA amplification by RT-PCR. This data is shown in Fig. 1.

Figure 1. Small RNA was amplified from 50 ng of a pool of RNAs from 4 different tissues with the SBI kit. The amplified cDNA was then tested for the presence of 12 small RNAs including 10 specific miRNAs by RT-PCR.  The number of PCR cycles was adjusted to provide similar amounts of PCR products.  Differences in cycle numbers between the different small RNAs were as high as 6 cycles.

Generation of Amplified Sense RNA by T7 IVT

To generate labeled hybridization targets for microRNA microarrays, the amplified cDNA can be used as template for T7-based In Vitro transcription (T7 IVT) in the presence of fluorescent labeled nucleotides by standard methods. An example of a T7 IVT experiment is shown in Fig. 4A and B.

Figure 4A. 50 ng of a pool of RNA from 4 different tissues was amplified with the SBI kit and purified. A small sample of the amplified cDNA was subjected to T7 IVT and then electrophoresed on a 3% agarose gel. The yield of amplified RNA was 22 μg.

Figure 4B. A small sample of the amplified sense RNA from Figure 4A was used as a template for T7 IVT. The amplified sense RNAs were then tested for the presence of 5 small non-coding RNAs, including 3 microRNAs (181a, 24, and 150) by RT-PCR. All 5 small RNAs were present in the amplified RNA.

List of Components

Each Global MicroRNA Amplification and Cloning Kit provides enough material to amplify small RNA from 10 different RNA samples (10 reactions).

Each kit contains the following components:

Kit Components

Size	Description
8μl	Ligase Cocktail (Blend of ligases)
70μl	Ligation Buffer
20μl	Control RNA (50 ng/μl)
1.2μl	RNase-free Water
10μl	3’ Adaptor
60μl	3’ Adaptor Primer
10μl	5’ Adaptor
50μl	5’ Adaptor Primer
8μl	Reverse Transcriptase
60μl	5X Reverse Transcriptase Buffer
20μl	Dithiothreitol (DTT)
50μl	dNTP Mix
150μl	10X PCR Buffer
35μl	PCR Polymerase (Proof-reading)
	Complete product manual included

Kit Storage Conditions
Store kit at -20° C